Efficient mechanisms for the clearance of lipopolysaccharide (LPS) from the circulation are critical to prevent excessive or unwanted inflammation, particularly during infection. The observation that elevated circulating LPS levels correlate with adverse outcome in critically ill sepsis patients suggests that normal clearance mechanisms become impaired or dysfunctional in severe sepsis. Therefore, defining the physiologic mechanisms for LPS clearance is essential to understanding how this process is altered in severe infection. Our previous work supports a central role for a novel Toll-like receptor (TLR) 4-dependent signaling complex on hepatocytes (HC) for the efficient uptake of LPS. This TLR4-dependent LPS uptake pathway in HC is distinct from the canonical TLR4-dependent pathway described in macrophages for the activation of innate immune mechanisms. We have also established the models to extend our work in vivo. Using both TLR4 bone marrow chimeric mice and novel cell specific TLR4-/- mouse strains developed in our lab, we have confirmed the requirement of TLR4 for the uptake of LPS by HC and that LPS uptake in KC is TLR4- independent. These observations lead us to propose that the efficient LPS uptake mechanisms in KC clear low levels of LPS intermittently released by the gastrointestinal tract, while TLR4-dependent uptake of LPS by HC contributes to uptake of higher LPS levels, or boluses of LPS, encountered during severe infection. Our objectives in this proposal are to fully test the hypothesis that a novel TLR4-signaling complex on HC contributes to HC LPS uptake and LPS clearance from the circulation during endotoxemia and sepsis. This work will challenge the paradigm that LPS clearance is mainly dependent on KC and mostly TLR4-independent. By fully characterizing the TLR4, CD11b/CD18-dependent signaling pathways involved in HC LPS uptake, we will define and functionally characterize a TLR4-dependent signaling pathway distinct from the canonical TLR4 pathway that leads to inflammatory mediator production. For part of this work, we will exploit a novel strategy using our recently developed TLR4-loxP mouse strains to selectively delete TLR4 from specific cell populations in the liver. Utilizing these mice, we will definitively establish the role of TLR4 on specific cell populations in LPS uptake, as well as LPS clearance, and we will explore the consequences of impaired LPS uptake in our models. We expect to show that HC clear LPS in the setting of endotoxemia and sepsis, to define the signaling mechanism leading to LPS uptake in HC, and to show that impairment of this mechanism leads to an exaggerated or sustained inflammatory response. By defining the pathways leading to TLR4-dependent LPS uptake, we expect to contribute to the understanding of how the process fails during severe infection. This, in turn, could lead to strategies that promote LPS clearance and improve outcomes in critically ill septic patients.